1. Field of Invention
The present invention relates to biotechnology, more particularly to a multi-target recombination gene and the application of its protein to prevent and cure Helicobacter pylori. 
2. Description of Related Arts
Helicobacter pylori (Hp) is an important pathogenic bacteria that was found in 1982. The study shows that this bacteria can not only cause gastritis and gastric ulcer, but also is closely relevant to the MALT lymphoma and gastric cancer. This bacteria is the only bacterial pathogens that is relevant to the human tumor announced by WHO. The recent study also discovers that the Hp is also closely relevant to the cardiovascular disease such as coronary disease. Hp can infect seniors, children and young adults, but the infection rate differs a lot in different countries and regions and differs according to different economic levels and living habits. The infection rate is about 50-80% among the general population in China, and it is increasing by 1-2% each year.
The urease gene of Hp was cloned in 1989, and the complete genome sequence of Hp was determined in 1997. Agnes etc analyze the urease by utilizing the shuttle clone technology, and confirm that a group of genes of encoding urease is located in the 4.2 kb DNA fragment, and there are 4 open reading frames (ORF), which are named ureA, ureB, ureC and ureD respectively. Further study shows that the ORF of ureC and ureD next to the ureA and ureB are ureI, ureE, ureF, ureG and ureH respectively. The length of the entire urease gene is 8 kb. In 1998, Stéphane of Institute Pasteur believes that the ureI gene is irrelevant to the urease of Hp, but is closely relevant to the gastric colonization, and is the necessary gene to the gastric colonization of Hp. In 2000, Rektorschek did comparative study by using genetic mutation technique and pointed that the ureI encodes the urea membrane channel protein of Hp. UreI gene transforms to 6 fragments of transmembrane protein UreI via in vitro transcription and translation. The protein is independent to the cell membrane of Hp, and protects the urease activity in the stomach acid environment where pH is lower than 4. The experiment shows that the intracellular urease can maintain activity without ureI protein when pH is higher than 4, but ureI is necessary gene to maintain urease activity when pH is lower than 4. In 2001, David L. Weeks believes that the UreI is the important channel protein for Hp to connect with the urease and gastric colonization. UreI has key function to the gastric colonization of Hp. There is a research that uses Xenopus oocytes as transgenic cell model to observe that the expression of ureI can promote the absorption of urea in an acid environment. However, the ureI mutant where the periplasmic histidine 123 is missing can not promote the absorption of urea. In the mean time, the urea transportation mediated by ureI is urea specific, passive, unsaturated and nonpolar, and non temperature-dependent. Weeks pointed out that the ureI is the H+ controlled urea channel of Hp, adjusts and controls the metabolism of intracellular urease, and is very important to the gastric colonization and survival of Hp. David R. Scott's study suggests that, with urea, ureI can stimulate the production of intracellular ammonia, but replacing urea with acetamide does not have the same result. Therefore, the function that ureI assists urease to resolve urea is specific. Furthermore, study also shows that UreI has four conservative histidine residues (H71, H123, H131, H193); three intracellular polypeptide rings has conservative zones (G166, K167, F168); the far end H193 (not H123) plays a decisive role for Hp to produce ammonia in the low pH environment; the third intracellular ring of UreI is important to the membrane channel activity of UreI. These illustrate the new molecule mechanism of gastric colonization of Hp.
A great amount of studies and clinical treatment experiment show that the infection and colonization of Hp is the primary cause of pathogenicity. When Hp is cleared, Gastritis and Gastric Ulcer get better. The pH value of human gastric mucosa from 2 to 4, and the pH value of gastric juice is around 2. Weeks' study shows that the extracellular urease of Hp is inactive when pH value is no more than 4.5, and can survive no more than 5 minutes when pH value is lower than 4.0. So why can Hp survive in the high acid environment of the stomach? Weeks and Scott believe that the urea channel UreI of Hp can take urea from outside of cell for intracellular urease to be resolved into NH3 and CO2. The ammonia cloud formed by the NH3 creates a cozy environment of hypoxia and weak acid for colonization, which becomes the necessary molecule for Hp colonization. However, there is no further report on the immunological characteristics of UreI molecule and whether it can be served as drug target to avoid Hp infection.
In 2000, Scott etc found that UreI is important to the activation of intracellular urease of Hp in the acid environment. In 2001, Week etc discovered that UreI is very important to the gastric colonization and pathogenicity of Hp through genetic mutation research. Skouloubris verified that UreI is irrelevant to the activity of urease via gene silencing technology such as RNAi, but is necessary to the colonization and proliferation of Hp in low pH value environment. In 2002, Mollenhauer proved that UreI is important to the colonization and the survival after colonization of Hp via experiment on Gerbillinae.
In 1998, Skouloubris etc verified that UreB is a subunit of urease activity, and is a necessary gene for colonization and proliferation of Hp in low pH value environment. In 2009, oral recombination Helicobacter pylori vaccine, whose main components is recombination UreB protein, is approved by the Chinese State Food and Drug Administration for first class new drug in April 2009, and enters stage of industrialization and clinical expansion and application. Though UreB is the acknowledged target of Hp vaccine, because of its many drawbacks, such as single gene target, prokaryotic expression, preparation purification, renaturation preservation, combine immunodominant antigen epitope in series so as to form multi-target recombination gene, which can be served as muti-target DNA vaccine and its corresponding recombinant protein vaccine or specific antibody product for preventing and curing Hp. Currently, there is still no report on biopharmacy medicine research of multi-target recombination gene and its protein for preventing and curing Hp.
Currently, from the current situation of Hp clinical treatment, the routine treatment for Hp infection is to use antibiotics or combined with antibiotics. However, though this broad-spectrum antibiotics treatment plays a certain role for removing Hp, this treatment may easily causes flora disorder and produces drug resistant strain, and is against the prevention and cure of Hp in the stomach and natural environment. Proton Pump Inhibitor (PPI) treatment can inhibit gastric acid secretion, but with the rebound of HP value in stomach, it creates a neutral environment for Hp colonization, and works to the advantage of Hp number increasing.
From the molecular interaction and biochemical mechanism related to Hp physiological metabolism, block the urea membrane channel by utilizing immune technique so as to block the biochemical reaction of urease resolving urea, and use Hp urease B subunit as target to make the intracellular and extracellular activity of resolving urea disappear at the same time, so that the Hp cannot survive and colonize in the gastric environment. The multi-target combination design by utilizing key molecule of Hp colonization can prevent and cure Hp infection and avoid the drawback of the antibiotics and PPI reagent in the clinical treatment. This biotechnology of polygene and multi-target immunodominant antigen epitope combination is the ideal way to prevent and cure Hp.